Fig. 7.

The SYCE1 core anti-parallel structure is stabilised in a tethered dimer construct. a A tethered dimer of SYCE1 core (amino acids 25–179) was generated through fusion of two sequences using either a long (GQTNPGTNPTG) or short (GQTNPG) linker. In a parallel coiled-coil configuration, this is predicted to generate a dimer of “tethered dimers” in series, whereas in an anti-parallel configuration, it is predicted to form a monomer of “tethered dimers.” b Far-UV circular dichroism (CD) spectra of SYCE1 core tethered dimers with short (black, solid) and long (black, dashed) linkers. Data were deconvoluted using the CDSSTR algorithm revealing helical content of 85% and 75%, respectively, with normalised r.m.s. deviation values of 0.010 and 0.001. SYCE1 core is shown in grey for comparison, exhibiting 65% helicity. c CD thermal denaturation of SYCE1 core tethered dimers with short (black, solid) and long (black, dashed) linkers, recording the CD helical signature at 222 nm between 5 and 95 °C, as % unfolded. Melting temperatures were estimated at 51 °C and 49 °C, respectively. SYCE1 core is shown in grey, with a melting temperature of 39 °C. d SEC-MALS analysis of SYCE1 core tethered dimers. The short linker and long linker constructs are monomers of “tethered dimers” of 38 kDa and 39 kDa, respectively (theoretical masses—37 kDa and 38 kDa); the SYCE1 core 36 kDa dimer is shown in grey. e SEC-SAXS scattering data for the SYCE1 core tethered dimer (short linker) construct, shown in black. A theoretical model of an anti-parallel SYCE1 dimer was generated from ideal coiled-coil and helical fragments, through CORAL rigid-body and linker modelling with fitting to experimental SAXS data (red; χ² = 1.48). Corresponding fit residuals are shown (inset). f SEC-SAXS P(r) distributions of SYCE1 core tethered dimer constructs with short (black, solid) and long (black, dashed) linkers. Maximum dimensions (Dmax) are indicated; SYCE1 core is shown in grey. g SAXS ab initio model of the SYCE1 core tethered dimer with short linker; an averaged model was generated from 25 independent DAMMIF runs with an NSD value of 0.710 (± 0.043) and reference model χ² value of 1.24. h SAXS CORAL model of the SYCE1 anti-parallel tethered dimer (short linker), generated through rigid-body and linker fitting to experimental data (χ² = 1.70)